flotation technique for detecting nematodes in the stool of dogs

זיהוי תולעים עגולות זואונוטיות בטכניקת צואה

Gastrointestinal parasites are one of the most common health problems that affect dogs1. Estimates suggest that there are ~700 million domestic dogs worldwide, and approximately 175 million may be categorized as free-roaming2. More than 60 parasite species are shared between dogs and humans, suggesting that dogs could be a source of infection for humans with these parasites3. Toxocara canis and Ancylostoma caninum are two parasitic species that infect dogs and, accidentally, human hosts. Currently, there are several studies about the locations where these helminths are able to survive and reproduce in Mexico. The prevalence of Toxocara in dogs varies from 0% to over 87% across the United States, Mexico, Central America, and the Caribbean4. Toxocara canis and Ancylostoma spp., as well as other parasitic species in dogs, have been previously reported in Mexico5,6,7,8,9,10,11,12,13&#160;(Table 1).
<table border="1" fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parasitic species</td>
			<td>Region</td>
			<td>Prevalence (%)</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td rowspan="4">Ancylostoma caninum</td>
			<td>Quer&#233;taro</td>
			<td>42.90</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Tabasco</td>
			<td>15.90</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Campeche</td>
			<td>35.7 &#8211; 42.9&#160;</td>
			<td>7</td>
		</tr>
		<tr>
			<td>Yucat&#225;n</td>
			<td>73.8</td>
			<td>8</td>
		</tr>
		<tr>
			<td rowspan="2">Babesia</td>
			<td>Morelos</td>
			<td>13.60</td>
			<td rowspan="2">9</td>
		</tr>
		<tr>
			<td>Veracruz</td>
			<td>10.00</td>
		</tr>
		<tr>
			<td>Coccidial oocysts</td>
			<td>Yucat&#225;n</td>
			<td>2.30</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Ctenocephalides</td>
			<td>Morelos</td>
			<td>30.3</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Dipylidium caninum</td>
			<td>Yucat&#225;n</td>
			<td>2.30</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Dirofilaria&#160;</td>
			<td>Yucat&#225;n</td>
			<td>7.0 &#8211; 8.3</td>
			<td>11</td>
		</tr>
		<tr>
			<td rowspan="2">Giardia</td>
			<td>Tabasco</td>
			<td>3.00</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Yucat&#225;n</td>
			<td>18.8</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Leishmania</td>
			<td>Chiapas</td>
			<td>19.00</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Tapeworms</td>
			<td>Baja California</td>
			<td>6.79</td>
			<td>13</td>
		</tr>
		<tr>
			<td rowspan="2">Toxocara canis</td>
			<td>Quer&#233;taro</td>
			<td>22.10</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Yucat&#225;n</td>
			<td>6.20</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Trichuris vulpis</td>
			<td>Yucat&#225;n</td>
			<td>25.40</td>
			<td>8</td>
		</tr>
		<tr>
			<td rowspan="6">Trypanosoma</td>
			<td>Jalisco</td>
			<td>8.10</td>
			<td rowspan="6">9</td>
		</tr>
		<tr>
			<td>Campeche</td>
			<td>7.60</td>
		</tr>
		<tr>
			<td>Chiapas</td>
			<td>4.5 &#8211; 42.8</td>
		</tr>
		<tr>
			<td>Quintana Roo</td>
			<td>20.1 &#8211; 21.3</td>
		</tr>
		<tr>
			<td>Toluca</td>
			<td>17.50</td>
		</tr>
		<tr>
			<td>Yucat&#225;n</td>
			<td>9.8 &#8211; 34</td>
		</tr>
	</tbody>
</table>
Table 1: Regional prevalence (%) of dog parasites in Mexico from 2001 to 2020. Findings from previous investigations conducted from 2001 to 2020 have enabled the identification of canine parasite distribution across several urban and rural settings in Mexico. These studies provide a deep understanding of epidemiological elements conducive to the persistence of canine parasites in different ecosystems, contributing to a comprehensive assessment of the zoonotic impact of some parasite species.
Life cycle stages of intestinal parasites, such as eggs, cysts, oocysts, or larvae can be found in stool samples. Thus, examination of fecal material provides valuable information about the parasites of an animal. The need for a method to detect Ancylostomidae eggs in human feces led to the use of the simple fecal smear in 1878, which was used for many years to detect gastrointestinal parasites but was considered not very sensitive. Thus, the need arose to develop better copromicroscopic methods14. More than 100 years have passed since the flotation technique for recovering and counting parasite eggs in fecal samples was first described15. Since then, several methods and variants of the flotation technique have been considered a standard for the detection of some parasites in their hosts.
For example, Lane described a method in 1924 involving the direct centrifugal flotation technique, which integrates centrifugation followed by floating the sediment in a saturated sodium chloride solution with SpG 1.2 in 1 g (Lane) or 10 g (Stoll&#39;s modification). The flotation technique was subsequently modified by using solutions with different SpG14. In 1939, Gordon and Whitlock reported the disadvantages of Stoll&#39;s technique due to interference from detritus in visualizing parasite eggs and developed the quantitative method known as McMaster16. In 1979, O&#39;Grady and Slocombe demonstrated that the specific gravity of the solution, timing, and mesh sizes of strainers affect the accuracy of egg detection using the flotation technique17. During the last decades, because several modifications have been made to the flotation technique, there is an urgent need for standardization of flotation methods. Currently, detection of canine helminth infections in the context of prevention of zoonotic parasites is required to apply appropriate anthelmintic treatments to limit environmental contamination with infectious stages of zoonotic nematodes18.
Among qualitative methods, the fecal flotation technique is widely used and accepted because it does not require much equipment, is simple, inexpensive, and reproducible; yet it has a major drawback in that it lacks sensitivity when the intensity of the infection is low19. The ability to reveal the presence of a greater number of parasitic elements such as eggs, oocysts, cysts, or nematode larvae is usually determined by the density of the solution20.
Previous reports have compared coproparasitological techniques for the detection of canine nematode eggs. With regard to the detection of motile protozoa, direct fecal smears are used; whereas sedimentation methods are useful for diagnosing heavy eggs of parasites such as trematodes21. One of the most widely used field-based diagnostic tests is the fecal smear method. However, the low level of sensitivity of this technique may be attributed to the fact that it does contain debris that interferes with the detection of parasite eggs. By incorporating a sieving step along with solutions that provide the proper SpG, the flotation method offers a clearer and less cluttered observation of ascarid and hookworm eggs. This leads to a more precise and efficient process for microscopic screening22. Likewise, simple flotation and direct centrifugal flotation techniques are very commonly utilized to recover parasite eggs and oocysts14. The classic flotation methods can be considered qualitative or quantitative depending on the use of a counting chamber such as the McMaster method15. Nonetheless, as the flotation technique has low sensitivity and focuses on the detection of parasites in the patent period, negative results should not be considered conclusive. However, the accuracy not only depends on the preservation procedure of fecal samples or the SpG of flotation solutions but also depends on the technical proficiency and experience in conducting fecal examinations of the user.
Consequently, other methods have been explored for the detection of canine parasites in feces. It has been generally recognized that one of the most widely used approaches for the diagnosis of intestinal helminth infections in dogs is the FLOTAC technique, a multivalent, sensitive, and accurate method that yields accurate and reliable results for the diagnosis of A. caninum in dogs when compared to a flotation protocol in a tube and the McMaster technique19,23. Sedimentation methods are useful for recovering fluke eggs, embryonated nematode eggs, and most tapeworm eggs, which cannot be recovered on the surface of a flotation solution because these structures do not float24. One method that has been proven to be superior to flotation/sedimentation techniques is the Modified Double Centrifugal Flotation method, as it enables the detection of cestode eggs in feces, is less time-consuming, separates Anoplocephala eggs from fecal debris, and decreases crystallization25. Moreover, this technique has been successfully used to detect ascarid eggs with high sensitivity26. Yet, some of these aforementioned techniques and centrifugal methods such as as the Ovassay, as opposed to the flotation protocol we propose in this study, require sample preservation in reagents such as formalin, commercial kits, sample processing under laboratory conditions, and the use of reagents such as zinc sulfate27&#160;which are expensive and require special disposal procedures to avoid environmental toxicity.
The use of techniques that increase the sensitivity of the flotation method by adding solutions with high SpG has been favored recently. However, it must be considered that the disadvantage of these solutions is the increase in debris in the final preparation and hence, the inaccurate detection of parasite eggs. In addition, the commercial availability of materials, reagents, cost, environmental impact issues, and difficulty of use of centrifugal methods affect the selection of a flotation technique14, which can be challenging in field conditions in contrast to the protocol that we present in this work. The preparation of the flotation solutions with table salt is advantageous over the utilization of sugar because under field conditions, sugar attracts insects such as wasps and bees and preparations become sticky. Further, solutions such as phenol, which is added to sugar solutions to avoid stickiness, or ZnSO4are complex to properly discard according to environmental protection guidelines and cannot be disposed of in the field; unlike a table salt solution.
The goal of this manuscript is to demonstrate the steps to detect T. canis and Ancylostoma spp. eggs in fecal samples using an adaptation of the simple flotation technique under field and laboratory conditions. Following the protocol herewith described and using a microscope with a backup battery, the diagnosis of these canine zoonotic parasites in rural and suburban areas is possible when no laboratory equipment and infrastructure are available. The simple flotation method described in this work can provide quick results and is a non-invasive and cost-effective technique for routine screening.

מחבר זרקור: בדיקה קופרופארסיטוסקופית של צואה של כלבים לשליטה ומניעה של מחלות טפילים זואונוטיות

שיטת הנפקה צואה פשוטה לאבחון נמטודות זואונוטיות בתנאי שדה ומעבדה

Currently, a countrywide geolocation study of the zoonotic parasites of dogs is being carried out to put further control and prevention strategies for these diseases. The existence of a wide variety of ecosystems in Mexico influences the frequency of parasite infections in dogs. Therefore, it is necessary to carry out field work and identify these parasites to be able to propose region-specific deworming guidelines according to the epidemiology of each parasite.The simple flotation technique is not sensitive enough to detect Cryptosporidium oocytes, Giardia cysts, or trichuriasis. We propose a concentration procedure for better coproparasitoscopic detection of these parasites. This technique can speed up and facilitate diagnosis of helminth infection in dogs in areas where no laboratory infrastructure or equipment are available.Findings and deworming recommendations can be prescribed immediately after each microscopic examination. This protocol can be helpful in conducting routine fecal examinations under field or laboratory conditions to estimate the frequencies of Toxocara canis and Ancylostoma infections in dogs.

טכניקת הנפקה לאיתור נמטודות בצואה של כלבים

כדי להתחיל, לאסוף את דגימות צואה מן הכלבים ולהכין את תמיסת מלח רווי בבקבוק של ליטר אחד. מניחים כשלושה גרם צואה בכוס פלסטיק. מוסיפים מיליליטר אחד מתמיסת המלח הרווי לכוס ומערבבים במשך דקה לקבלת משחה.מוסיפים 100 מיליליטר של תמיסת מלח רווי לכוס. העבירו את המתלה דרך מסננת פלסטיק לכוס פלסטיק טרייה כדי להסיר חלקיקים גסים. הניחו למתלים לעמוד למשך 15 עד 20 דקות.חממו לולאת חיסון למשך שנייה אחת כדי לוודא שהיא נקייה מביציות, ציסטות או ביציות, ותנו לה להתקרר במשך חמש שניות. קח בערך שלוש טיפות של תרחיף הצואה מפני השטח עם לולאת החיסון. הניחו שלוש טיפות נפרדות על מגלשת זכוכית.התבוננו בכל טיפה מתחת למיקרוסקופ עם מטרה של פי 10. הניחו פתק כיסוי על הדגימה כדי להמחיש את הדגימה בהגדלה של פי 40. תמונות מיקרוסקופ האור בהגדלה של פי 40 הראו נוכחות של Toxocara canis וביצי מין Ancylostoma.

Research

JoVE Journal

Immunology and Infection

Flotation Technique for Detecting Nematodes in the Stool of Dogs

A Simple Fecal Flotation Method for Diagnosing Zoonotic Nematodes Under Field and Laboratory Conditions

Diagnosis of canine parasites with zoonotic potential such as Toxocara canis and Ancylostoma caninum under field conditions is usually difficult due to ...

Collecting Fecal Samples of Dogs for Nematode Detection

איסוף דגימות צואה של כלבים לאיתור נמטודות 